Backflow prevention for high pressure gradient systems

ABSTRACT

Gradient performance with high pressure gradient solvent delivery system is optimized by approximation of infinite stroke volume of high pressure pumps by the addition of pulse dampening with backflow prevention to each high pressure pump. The backflow prevention adds sufficient minimum flow resistance, thereby enhancing the performance of the pulse dampening over a wider range of flow rates resulting in consistent gradient performance.

FIELD OF THE INVENTION

The present invention relates to liquid chromatography instrumentationand solvent delivery systems, and more particularly to a method andapparatus for control of chromatographic pumping systems.

BACKGROUND OF THE INVENTION

High-pressure liquid chromatography (HPLC) solvent delivery systems areused to source single-component liquids or mixtures of liquids (bothknown as “mobile phase”) at pressures which can range from substantiallyatmospheric pressure to pressures on the order of ten thousand poundsper square inch. These pressures are required to force the mobile phasethrough the fluid passageways of a stationary phase support, whereseparation of dissolved analytes can occur. The stationary phase supportmay comprise a packed bed of particles, a membrane or collection ofmembranes, a porous monolithic bed, or an open tube. Often, analyticalconditions require the mobile phase composition to change over thecourse of the analysis (this mode being termed “gradient elution”). Ingradient elution, the viscosity of the mobile phase may change and thepressure necessary to maintain the required volumetric flow rate willchange accordingly.

In liquid chromatography, the choice of an appropriate separationstrategy (including hardware, software, and chemistry) results in theseparation of an injected sample mixture into its components, whichelute from the column in reasonably distinct zones or “bands”. As thesebands pass through a detector, their presence can be monitored and adetector output (usually in the form of an electrical signal) can beproduced. The pattern of analyte concentration within the eluting bands,which can be represented by means of a time-varying electrical signal,gives rise to the nomenclature of a “chromatographic peak”. Peaks may becharacterized with respect to their “retention time”, which is the timeat which the center of the band transits the detector, relative to thetime of injection (i.e. time-of-injection is equal to zero). In manyapplications, the retention time of a peak is used to infer the identityof the eluting analyte, based upon related analyses with standards andcalibrants. The retention time for a peak is strongly influenced by themobile phase composition, and by the accumulated volume of mobile phasewhich has passed over the stationary phase.

The utility of chromatography relies heavily on run-to-runreproducibility, such that standards or calibrants can be analyzed inone set of runs, followed by test samples or unknowns, followed by morestandards, in order that confidence can be had in the resulting data.Known pumping systems exhibit some non-ideal characteristics whichresult in diminished separation performance and diminished run-to-runreproducibility. Among the non-ideal pump characteristics exhibited inknown pumping systems are, generally, fluctuations in solventcomposition and/or fluctuations in volumetric flow rate.

Volumetric flow fluctuations present in known HPLC pumping systemsdisadvantageously cause retention time(s) to vary for a given analyte.That is, the amount of time that an analyte is retained in thestationary phase fluctuates undesirably as a function of the undesirablevolumetric flow fluctuations. This creates difficulties in inferring theidentity of a sample from the retention behavior of the components.Volumetric flow fluctuations from individual pumps can result influctuations in solvent composition when the output of multiple pumps issummed to provide a solvent composition.

Fluctuations in solvent composition present in known HPLC pumpingsystems can disadvantageously result in interactions with the system'sanalyte detector and produce perturbations which are detected as if theyarose from the presence of a sample. In effect, an interference signalis generated. This interference signal is summed with the actual signalattributable to the analyte, producing errors when the quantity of anunknown sample is calculated from the area of the eluting sample peak.

The prior art is replete with techniques and instrument implementationsaimed at controlling solvent delivery and minimizing perturbations inthe output of delivery systems for analytical instrumentation. Myriadpump configurations are known which deliver fluid at high pressure foruse in applications such as liquid chromatography. Known pumps, such asone disclosed in U.S. Pat. No. 4,883,409 (“the '409 patent”) incorporateat least one plunger or piston which is reciprocated within a pumpchamber into which fluid is introduced. A controlled reciprocationfrequency and stroke length of the plunger within the pump chamberdetermines the flow rate of fluid output from the pump. However, theassembly for driving the plunger is an elaborate combination of elementsthat can introduce undesirable motion in the plunger as it is driven,which motion makes it difficult to precisely control the solventdelivery system output and results in what is termed “noise” ordetectable perturbations in a chromatographic baseline. Much of thisnoise does not result from random statistical variation in the system,rather much of it is a function of a mechanical “signature” of the pump.Mechanical signature is correlated to mechanically related phenomenasuch as anomalies in ball and screw drives, gears, and/or othercomponents used in the pump to effect the linear motion that drives thepiston(s), or it is related to higher level processes or physicalphenomena such as the onset or completion of solvent compression, or theonset of solvent delivery from the pump chamber.

Typical systems known for delivery of liquids in liquid chromatographyapplications, such as disclosed in the '409 patent and further in U.S.Pat. No. 5,393,434, implement dual piston pumps having twointerconnected pump heads each with a reciprocating plunger. Theplungers are driven with a predetermined phase difference in order tominimize output flow variations. Piston stroke length and strokefrequency can be independently adjusted when the pistons areindependently, synchronously driven. Precompression can be effected ineach pump cylinder in any given pump cycle to compensate for varyingfluid compressibilities in an effort to maintain a substantiallyconstant system pressure and output flow rate.

There are two widely used means to create gradient HPLC pumps. Thesolvents can be blended on the intake side of the pump. This is known inthe art as low pressure gradient mixing. The alternative is the use ofso-called high pressure gradient systems in which each individualsolvent is delivered by a separate pump.

The fundamental scalar of all forms of gradient chromatography is thevoid volume of the separation column. The void volume of an HPLC columnis the sum of the inter and intra particle volumes of the column thatare filled with mobile phase. The void volume is the minimum volumerequired to elute an unretained solute. The gradient delay volume is thevolume of the mobile phase delivered from the time the gradient isinitiated to when the change in composition first arrives at the column.The delay volume is the volumetric overhead of the gradient solventdelivery system; it adds to the time required to complete the separationand to prepare the column for the next injection. The delay volumeshould be minimized and ideally should be no more than two times largerthan the void volume of the column.

When two or more high pressure pumps are combined to form a gradientsolvent delivery system, their outputs are combined with the resultingpossibility that there can be fluidic cross talk between the highpressure pumps during their individual piston crossovers. One priorapproach to avoid fluidic cross talk has been the use of pulse dampenerswithin the gradient solvent delivery system as shown in FIG. 1.

When individual pulse dampeners are placed up-stream from where outputof the solvents meet and the total flow is small relative to the volumeof the pulse dampeners, there will be significant crosstalk between thepumps since the two pumps are not synchronous in their respective pistoncrossovers. This crosstalk occurs because the fluid contained in the offline pulse dampener can be compressed making it the low impedance pathfor the on-line pump. As such, this up-stream placement of the pulsedampeners results in a compromised flow rate and composition. The resultof this fluidic crosstalk is shown in FIG. 2, which plots the deliveryof a gradient marker from a solvent delivery system configured as shownin FIG. 1 at a low flow rate. As shown in FIG. 2, no gradient deliveriesare identical and none correspond to the programmed gradient. Thisresults in unsatisfactory and unpredictable separations which cannot bereproduced.

In an alternative prior art approach, a capillary restrictor is used togenerate backpressure to energize the pulse dampeners. A capillary offixed length and internal diameter provides sufficient backpressure torestrict, but unfortunately not prevent backflow, over narrow ranges offlow rates.

A further approach to the use of pulse dampeners is to position a pulsedampener downstream from the common mixing tee. While this approach isuseful in gradient systems having large volumes, smaller scale volumesare problematic. The positioning of a pulse dampener after the commonmixing tee greatly increases the delay volume within the gradientsystems. Pulse dampeners are scaled to a specific and limited flow raterange as they typically combine resistance to flow and a captivecapacitive volume of the mobile phase. The requirements of effectivepulse dampening and minimizing delay volume will conflict as the scaleof the HPLC system with respect to column volume and volumetric flowrate is reduced.

SUMMARY OF THE INVENTION

The present invention provides an improved method and apparatus forimproving the compositional accuracy of high pressure gradient pumps forHPLC by approximation of infinite stroke volume with backflow preventedpulse dampening. The backflow prevention, according to the invention,adds sufficient minimum flow resistance thereby enhancing theperformance of the pulse dampening over a wider range of flow ratesresulting in consistent gradient performance.

According to the invention, pulse dampeners in conjunction with backflow preventors, which may be embodied as check valves or in-lineback-pressure regulators, ensure that the stored mobile phase iscompressed during the delivery cycle. When a backflow preventor with afixed minimum flow resistance is used, the effectiveness of the pulsedampener becomes substantially independent of the flow rate. The use ofbackflow preventors within the gradient system ensures that the storedmobile phase is compressed and has mechanical energy to return to thesystem at piston crossover.

The backflow preventors further ensure that the outlet check valves ofthe respective pump heads will experience sufficient backpressureallowing for their proper functioning. This sufficient backpressure isparticularly helpful in systems having low flow rates when thebackpressure generated by the column and tubing is limited.Additionally, backpressure allows the primary check valves of individualpumps to operate more consistently as the resulting backpressure ensuresproper seating of the outlet check valve on the pump head that is offline.

The proper placement of a backflow preventor according to the inventionreduces fluidic cross talk, optimizes the performance of in-line pulsedampers and enhances the performance of the high pressure pumps as shownin FIG. 3, which plots the delivery of a gradient marker from a solventdelivery system configured with backflow prevention according to theinvention. As depicted in FIG. 3, the gradient deliveries are identicaland correspond to the programmed gradient.

Advantageously, individual pumps deliver smooth flow by the addition ofsuitable pulse dampeners with the further use of backflow preventorsthat prevent fluidic cross talk between the two mobile phases. Because apulse dampener is the fluidic equivalent of a low pass filter, when asmall stroke volume is combined with a pulse dampener, the crossoverperturbations occur at frequencies that are strongly attenuated. Thedifferences between individual pump heads are effectively averaged bythe use of pulse dampeners. Thus, the use of small stoke volumes withefficient pulse dampening provide for uniform blending of solvents inhigh pressure gradient systems.

In an alternative illustrative embodiment a capillary restrictor is usedto generate backpressure to energize the pulse dampener. A capillary offixed length and internal diameter provides sufficient backpressure overa certain range of flow rates. The use of a capillary restrictor inseries with a check valve can be used for systems having consistent flowrates.

In a further alternative illustrative embodiment the check valves areincorporated into a mixing tee. This incorporation decreases the volumeof the mobile phase within the gradient system and therefore decreasesthe delay volume of the gradient system.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments, taken in conjunction with the accompanyingdrawings in which:

FIG. 1 is a schematic of a standard high pressure gradient pump (priorart);

FIG. 2 demonstrates problems with fluidic crosstalk in high pressuregradient systems (prior art);

FIG. 3 illustrates the effect of pump crossover on solvent compositionwithout backflow prevention (prior art);

FIG. 4 demonstrates the use of pulse dampers with the addition ofin-line check valves according to the invention;

FIG. 5 illustrates the cumulative effect of pump crossover on solventcomposition (prior art);

FIG. 6 is a schematic of a high pressure gradient pumping systemaccording to the invention;

FIG. 7 is a schematic of a mixing tee having integrated check valves;and

FIG. 8 is a schematic of a mixing tee having integrated check valves andbackpressure regulators.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure will be described in detail with respect tochromatographic applications with the understanding that embodiments ofthe present invention are directed to industrial and process controlapplications as well.

As shown in FIG. 4 the effect of pump crossover on solvent compositionis illustrated. Within this illustration, the total flow is 1 mL/min. Afirst pump delivers ninety percent of the flow or approximately 900μL/min. A second pump delivers ten percent of the flow or approximately100 μL/min. The stroke volume is approximately 100 μL for both pumps.There are nine crossovers of the first pump to one crossover of thesecond pump to provide the desired composition. When the first pumpcrosses over there is a deficit in the first solvent of about 23 percentin flow rate and the composition is momentarily enriched in the secondsolvent. This deficit is illustrated by a first curve 301. A secondcurve 302 shows the effect of reducing the magnitude of the flow ratedeficit from about 23 percent loss of flow at crossover to 10 percentloss of flow by the use of limited pulse dampening. The compositionalperturbations or “noise” is reduced from about ±3 percent of the secondsolvent delivered to about ±1 percent of the second solvent delivered.Further pulse dampening according to the invention would further reducethe compositional noise.

The effect of this compositional noise on the retention times of analytepeaks is strongly dependent upon the degree of retention of the analyteand is expressed in its k-prime (k′) value which is the number of columnvolumes required to elute the analyte from the column. The k′ value iscomputed from the following formula:

k′=(V_(r)−V_(o))/V_(o)  (Formula I)

where V_(r)=retention volume and V_(o)=column void volume.

When k′ is small, variations in mobile phase composition have littleeffect on retention volume, however when k′ is large small variations inmobile phase composition have a large effect on retention volume sincek′ is exponentially proportional to the percent of the second solventdelivered.

The cumulative effect of pump crossovers on the percent of the secondsolvent delivered is illustrated in FIG. 5. The cumulative error in thepercent of the second solvent is strongly coupled to the magnitude ofthe gradient pulse. The resulting variance in retention times will bestrongly coupled with the degree of pulsation and the mixing requirementensuring a more uniform composition is directly coupled to theinstantaneous and the cumulative errors in the percent of the secondsolvent. When the pulsations are reduced according to the invention thecomposition becomes inherently more uniform and requires a smallervolume to ensure its uniformity.

Turning to FIG. 6, an illustrative embodiment of the instant inventionis a high pressure gradient system in which each individual solvent isdelivered by a separate pump. This illustrative embodiment has a firstsolvent delivery line 101 and a second solvent delivery line 103. Afirst solvent is delivered to a first pump 105 within the first solventdelivery line 101 via a fluidic tee 104. The first pump 105 has a firstpiston 107 and a second piston 109. In this illustrative embodiment thefirst pump 105 is a Waters model HPLC pump 515, made by WatersCorporation of Milford Mass., which is a fluidic pump having a fixedstroke length. It is contemplated within the scope of this inventionthat other pumps known in the art may be used.

The first solvent is delivered via the first pump 105 to a prime valve111, such as Waters P/N WAT 207085, Waters Corporation, Milford, Mass.,which also acts as a fluidic tee receiving the output from the firstpiston 107 and the second piston 109. The first solvent is delivered toa first pulse dampener 112. The first pulse dampener 112, which in thisillustrative embodiment is a Waters High Pressure Filter, P/N WAT207072,Waters Corporation, Milford, Mass., is a fluidic low pass filter thatminimizes flow rate perturbation within the first solvent delivery line.It is contemplated within the scope of this invention that other pulsedampeners known in the art may be used.

The first solvent is pumped through the first pulse dampener 112 and isdelivered to a first backflow preventor 114. The first backflowpreventor 114, which in this illustrative embodiment is an UpchurchModel U-609, Upchurch Scientific, Oak Harbor, Wash., has a knownresistance to flow forces that causes a load onto the first pulsedampener 112 ensuring consistent operation of the first pulse dampener112. This resistance to flow can range from about 0 to 2,000 psi, and inthis first illustrative embodiment the resistance is approximately 250psi.

The first backflow preventor 114 is in fluid communication with a commonmixing tee 116 that directs the first solvent through a pressuretransducer 1 18 and into a vent valve 119, such as Rheodyne 7033,Rheodyne, LP., Rohnert Park, Calif. The vent valve 119 directs the firstsolvent to an injector and a chromatography column 120.

A second solvent is delivered to a second pump 122 within the secondsolvent delivery line 103 via a second fluidic tee 124. The second pump122 has a first piston 124 and a second piston 126. In this illustrativeembodiment the second pump 122 is a Waters model 515 HPLC pump, WatersCorporation Milford Mass., which is a fluidic pump having a fixed strokelength. It is contemplated within the scope of this invention that otherpumps known in the art may be used.

The second solvent is delivered via the second pump 122 to second primevalve 128, such as Waters P/N WAT 207085, Waters Corporation, Milford,Mass., which also acts as a fluidic tee receiving the output from thefirst piston 124 and the second piston 126. The second solvent isdelivered to a second pulse dampener 130. The second pulse dampener 130provides a fluidic low pass filter that minimizes flow rateperturbations within the second solvent delivery line. The secondsolvent is pumped to a second backflow preventor 132. The secondbackflow preventor 132 has a known resistance to flow forces that causesa pressure load onto the second pulse dampener 130 ensuring consistentoperation of the second pulse dampener 130. This resistance to flow canrange from 0 to 2000 psi, and in this first illustrative embodiment theresistance is approximately 250 psi.

The second backflow preventor 132 is in fluid communication with thecommon mixing tee 116 that directs the first solvent through thepressure transducer 118 and into the vent valve 119 and the secondsolvent 102 through the pressure transducer 118 and into the vent valve119. The vent valve 119, such as a Rheodyne 7033, Rheodyne, LP., RohnertPark, Calif., directs the first solvent 101 and the second solvent 102to the chromatography column 120.

In an alternative embodiment of the invention the first backflowpreventor and the second backflow preventor are incorporated into thestructure of the mixing tee to minimize system volume. As illustrated inFIG. 7 the common mixing tee 201 has a first backflow preventor 203 anda second backflow preventor 205 incorporated into the structure of themixing tee 201. The mixing tee 201 has a first inlet port 207 in whichthe first backflow preventor 203 is incorporated, a second inlet port215 in which the second backflow preventor 205 is incorporated and anoutlet port 214 in which fluid flow from the first inlet port 207 andthe second inlet port 215 are directed.

The first backflow preventor 203 has a first ball bearing 21 1 housedwithin a first check valve body 220. The first ball bearing 211 isseated in a first check valve seat 213. The first ball bearing 211 isfabricated from materials that are inert to system solvents such assapphire and ceramic or the like. The first ball bearing 211 is encasedin a first check valve cartridge housing component 221 in a mannerallowing only forward fluid flow. The first check valve cartridgehousing component 221 is comprised of a top part 222 and a base part224, which forms the first check valve seat 213.

The second backflow preventor 205 has a second ball bearing 217 housedwithin a second check valve body 223. The second ball bearing 217 isseated in a second check valve seat 218. The second ball bearing 217 isfabricated from materials that are inert to system solvents such assapphire and ceramic or the like. The second ball bearing 217 is encasedin a second check valve cartridge housing component 227 in a manner onlyallowing forward fluid flow. The second check valve cartridge housingcomponent 227 is comprised of a top part 229 and a base part 226 whichforms the second check valve seat 218.

In a further alternative embodiment of the invention the first backflowpreventor and the second backflow preventor are incorporated into thestructure of the mixing tee to minimize system volume. As illustrated inFIG. 8 the common mixing tee 301 has a first backflow preventor 303 anda second backflow preventor 305 incorporated into the structure of themixing tee 301. The mixing tee 301 has a first inlet port 307 in whichthe first backflow preventor 303 is incorporated, a second inlet port315 in which the second backflow preventor 305 is incorporated and anoutlet port 314 in which fluid flow from the first inlet port 307 andthe second inlet port 315 are directed.

The first backflow preventor 303 has a coil spring 309 that appliespressure to a first actuator 311. The first actuator 311 is seated intoa first valve opening 313. The selected coil spring 309 provides acertain resistance to flow by exerting pressure against the firstactuator thereby sealing the first valve opening 313 until theresistance to flow is exceeded.

The second backflow preventor 305 has a coil spring 316 that appliespressure to a second actuator 317. The second actuator 317 is seatedinto a second valve opening 318. Again, the selected coil spring 316provides a certain resistance to flow by exerting pressure against thesecond actuator 317 thereby sealing the second valve opening 318 untilthe resistance to flow is exceeded.

In a further alternative embodiment the pulse dampeners within thefluidic solvent delivery lines are configured from a section ofcapillary tubing whose length and diameter are optimized to provide thenecessary volume within the capillary tubing to minimize flow rateperturbations.

Although the chromatography pumping system described in the illustrativeembodiment herein is configured to accommodate two separate solventsources it should be appreciated that multiple or single solventdelivery systems as are known in the art can be implemented.

Although the chromatography pumping system described in the illustrativeembodiment herein is configured having traditional actuator and springbackflow preventors it should be appreciated that other backflowpreventors that are known in the art can be used.

The foregoing describes specific embodiments of the inventive method andapparatus. The present disclosure is not limited in scope by theillustrative embodiments described, which are intended as specificillustrations of individual aspects of the disclosure. Equivalentmethods and components are within the scope of the disclosure. Indeed,the instant disclosure permits various and further modifications to theIllustrative embodiments, which will become apparent to those skilled inthe art. Such modifications are intended to fall within the scope of theappended claims.

What is claimed is:
 1. A high pressure liquid chromatography apparatuscomprising: a first set of pumps and a second set of pumps said firstset of pumps being in fluid communication to a first purge valve andsaid second set of pumps being in fluid communication to a second purgevalve; a first pulse dampener and a second pulse dampener said firstpulse dampener being in fluid communication with a first backflowpreventor and said second pulse dampener being in fluid communication tosecond backflow preventor; a mixing tee said mixing tee being in fluidcommunication with said backflow preventors and being in fluidcommunication with a vent valve said vent valve being in fluidcommunication with a chromatography column.
 2. The apparatus forbackflow prevention according to claim 1 wherein said backflowpreventors have backpressure regulators having a selected fixedresistance said selected fixed resistance ensuring that said pulsedampeners function efficiently with consistent performance of primarycheck valves of said pumps.
 3. The apparatus for backflow preventionaccording to claim 1 wherein said backflow preventors improve accuracyof compositional delivery.
 4. The apparatus for backflow preventionaccording to claim 1 wherein said backflow preventors allow accuratecompositional delivery at flow rates substantially equivalent todisplaced volumes of each pump chamber of said pumps.
 5. The apparatusfor backflow prevention according to claim 1 wherein said backflowregulators are incorporated in said mixing tee to reduce delay volume.6. The apparatus of backflow prevention according to claim 1 whereinsaid incorporated backflow preventors have backpressure regulators. 7.The apparatus of backflow prevention according to claim 1 wherein saidbackflow preventors allow accurate compositional delivery at flow ratessubstantially less than the volumes of said pulse dampeners.
 8. Theapparatus for backflow prevention according to claim 1 wherein saidpulse dampeners reduce pump pulsations thereby reducing volumetricrequirement for effective solvent mixing allowing for the use of highpressure gradient systems in smaller volume chromatography columns.
 9. Ahigh pressure liquid chromatography comprising: a first set of pumpswithin a first solvent delivery line and a second set of pumps within asecond solvent delivery line said first set of pumps being in fluidcommunication with a first solvent reservoir and said second set ofpumps being in fluid communication with a second solvent reservoir;means for pulse dampening so that flow rate perturbations produce bysaid first set of pumps and said second set of pumps are reduced; andmeans for backflow prevention so that fluidic cross talk is eliminatedbetween said solvent delivery lines.
 10. The apparatus according toclaim 9 further comprising means for backpressure regulation.